Apoaequorin is a single polypeptide chain protein which can be isolated from the luminous jellyfish Aequorea victoria. When this protein contains one molecule of coelenterate luciferin bound noncovalently to it, it is known as aequorin. Aequorin is oxidized in the presence of calcium ions to produce visible light. Once light is produced, the spent protein (apoaequorin) can be purified from the oxidized luciferin and subsequently recharged using natural or synthetic luciferin under appropriate conditions. The addition of calcium ions to the recharged aequorin will again result in the production of light. Apoaequorin can therefore be used in various chemical and biochemical assays as a marker.
Natural apoaequorin is not a single compound but rather represents a mixture of microheterogeneous molecular species. When pure natural aequorin, representing that of many thousands of individual Aequorea, is subjected to electrophoresis (O. Gabriel, Methods Enzymol. (1971)22:565-578) in alkaline buffers under nondenaturing conditions, at least six distinct bands of blue luminescence are visible when the gel is immersed in 0.1M CaCl.sub.2. This observation agrees with that of J. R. Blinks et al. (Fed. Proc. (1975) 34:474) who observed as many as twelve luminescent bands after the isoelectric focusing of a similar extract. Blinks et al. observed more species because isoelectric focusing is capable of higher resolution than is electrophoresis. However, none of the bands was ever isolated as a pure polypeptide.
Furthermore, it is difficult to produce sufficient aequorin or apoaequorin from jellyfish or other natural sources to provide the amounts necessary for use in bioluminescence assays. Accordingly, an improved means for producing apoaequorin in sufficient quantities for commercial utilization is greatly needed.
Known molecular biology and recombinant DNA techniques permit one skilled in the art to synthesize a protein or peptide normally made by another organism. These techniques make use of a fundamental relationship which exists in all living organisms between the genetic material, usually DNA, and the proteins synthesized by the organism. This relationship is such that the amino acid sequence of the protein is reflected in the nucleotide sequence of the DNA. There are one or more trinucleotide sequence groups specifically related to each of the twenty amino acids most commonly occurring in proteins. As a consequence, the amino acid sequence of every protein or peptide is reflected by a corresponding nucleotide sequence, according to a well understood relationship. Furthermore, this sequence of nucleotides can, in principle, be translated by any living organism.
In its basic outline, a method of endowing a microorganism with the ability to synthesize a new protein involves three general steps: (1) isolation and purification (or chemical synthesis) of the specific gene or nucleotide sequence containing the genetically coded information for the amino acid sequence of the desired protein, (2) recombination of the isolated nucleotide sequence with an appropriate vector, typically the DNA of a bacteriophage or plasmid, and (3) transfer of the vector to the appropriate microorganism and selection of a strain of the recipient microorganism containing the desired genotype.
The symbols and abbreviations used herein are set forth in the following table.
TABLE 1 ______________________________________ DNA, deoxyribonucleic acid A-Adenine RNA, ribonucleic acid T-Thymine cDNA, complementary DNA G-Guanine (enzymatically synthesized C-Cytosine from an mRNA sequence) U-Uracil mRNA, messenger RNA Tris-2-Amino-2 hydroxymethyl 1,3-propanediol dATP, deoxyadenosine triphosphate dGTP, deoxyguanosine triphosphate dCTP, deoxycytidine triphosphate EDTA, ethylene diamine tetra-acetic acid TCA - Trichloroacetic acid dTTP - thymidine triphosphate ATP - adenosine triphosphate ______________________________________
Restriction endonucleases are enzymes capable of hydrolyzing phosphodiester bonds in DNA, thereby creating a break in the continuity of the DNA strand. The principal feature of a restriction enzyme is that its hydrolytic action is exerted only at a point where a specific nucleotide sequence occurs. Such a sequence is termed the restriction site for the restriction endonuclease. When acting on double-stranded DNA, some restriction endonucleases hydrolyze the phosphodiester bonds on both strands at the same point, producing blunt ends. Others catalyze hydrolysis of bonds separated by a few nucleotides from each other, producing free single stranded regions at each end of the cleaved molecule. Such single-stranded ends are self-complementary, hence cohesive, and may be used to rejoin the hydrolyzed DNA. Since any DNA susceptible to cleavage by such an enzyme must contain the same recognition site, the same cohesive ends will be produced, so that it is possible to join heterogeneous sequences of DNA which have been treated with restriction endonuclease to other sequences similarly treated.
Tsuji, et al., (1986) Proc. Natl. Acad. Sci. USA 83:8107-8111, disclose specific mutations in the aequorin amino acid sequence. However, Tsuji, et al. conducted their studies on the 189 amino acid fragment of aequorin, which is missing the first seven amino acids of the protein. Their experiments were designed to investigate the role of putative Ca.sup.2+ binding sites in the protein, the three cysteines, and a histidine in the hydrophobic region. Each of the routants generated resulted in aequorin having activity below that of fully regenerated unmodified 189 amino acid aequorin fragment. See Tsuji, et al. Table 2. What is needed, however, is modified aequorin proteins having greater activity than naturally occurring or unmodified recombinantly produced aequorin.